We seek to develop DNA-binding pyrrole-imidazole (Py-Im) polyamides as therapeutics for the neuromuscular disorder myotonic dystrophy type 1 (DM1). Py-Im polyamides are a class of synthetic minor groove-binding ligands that can be programmed to bind predetermined DNA sequences, such as the CTG/CAG triplet repeats that are causative of DM1. Long repeats in the DMPK gene are transcribed into a toxic gain-of-function poly(CUG) RNA that sequesters essential mRNA splicing factors, and may also be translated into toxic protein species. We have generated human induced pluripotent stem cell (hiPSC) clones of identical genetic background but different CTG/CAG triplet-repeat size from DM1 patient fibroblasts by transcription factor reprogramming, and these cells have been differentiated into the relevant cell types in the human disease DM1, such as neurons, cardiomyocytes and myocytes. We have made the important observation that a specific Py-Im polyamide targeting the CTG/CAG triplet-repeats is able to virtually abolish nuclear foci in patient cells. This molecule is without effect on genes that harbor short, non-pathogenic CTG/CAG repeats but shows a decrease in DMPK mRNA in DM1 cells, consistent with down regulation of the pathogenic allele. We will establish the therapeutic potential of the polyamides with the following specific aims: (1) We will fully characterize DM1 and control hiPSC-derived neurons, cardiomyocytes and myocytes, for studies of polyamide efficacy. DM1 signatures will be investigated, including CUG nuclear RNA foci formation and defects in RNA spicing. The efficacy of the Py-Im polyamide in these differentiated cells will be assessed by monitoring CUG RNA foci, transcription of the pathogenic DMPK allele and restoration of normal splicing patterns of genes that are affected in DM1 cells. Improvements in cellular uptake and compound stability will be achieved by synthetic chemistry efforts. (2) Synthetic methods will be optimized for preparation of sufficient quantities of the active compound for animal studies. The pharmacokinetics, maximum tolerated dose and tissue distribution of the most efficacious molecules will first be determined in normal mice. (3) Efficacy in a mouse model that expresses a long CUG RNA will be explored with Dr. Charles Thornton of the University of Rochester. Manifestations of the disease, such as myotonia, as well as biochemical markers (splicing defects and nuclear RNA foci) in affected muscle will be monitored.